Nine microsatellite loci were developed for Octopus mimus, a cephalopod of commercial importance for artisanal fishermen. Genetic variation at these loci was examined in samples from Clavelito, a Benthic Resources Management Area (AMERB, in Spanish). All nine loci were highly polymorphic, with the number of alleles per locus ranging from 4 to 28 and the expected heterozygosity ranging from 0.651 to 0.946. These markers will be useful to address issues of population genetics, ecology, conservation and fisheries management related to that species.

Key words: Population genetics, AMERBs, cephalopod

INTRODUCTION

To evaluate how genetic variability is distributed it is necessary to use polymorphic molecular markers. In the last few years, microsatellites have been some of the most used molecular markers for determining genetic variability and genetic structure levels (Barbará et al. 2008). These codominant markers are higly polimorphic, with motifs of 1 to 6 nucleotides in length, organized in tandems, widely distributed along the genome (Tautz 1989).

In cephalopods as in other marine groups, few studies have been devoted to identify and characterize polymorphic microsatellite loci. This is unusual, due to the low genetic diversity that has been observed using others molecular markers in cephalopods (i.e., Allozymes: Brierley et al. 1995, Triantafillos et al. 2004 and mitochondrial DNA: Oosthuizen et al. 2004).

Octopus mimus (Gould, 1852), is a member of Octopodidae Family, it inhabits rocky shore systems in the Southeast Pacific from northern Perú to central Chile. Biological traits such as separate sexes, internal fertilization and a semelparous reproductive strategy (Leite et al. 2008) make this species a good model to answer questions about genetic connectivity. Most studies in the Genus Octopus have focused on determining the phylogeny (e.g., Warnke 2004) and only a few focus on their population biology. Even a smaller number of these studies used microsatellite loci as a tool for determining genetic structure (e.g., Greatorex et al. 2000, Cabranes et al. 2008, Doubleday et al. 2009). Up until now, there is no information about O. mimus population genetics. In this paper, we describe the isolation, characterization and utility of nine microsatellite loci for O. mimus in one Chilean resource management area.

MATERIALS AND METHODS

High molecular weight DNA was extracted from arm tissue of 20 individuals from both sexes using a salting-out protocol (Jowett 1986). The genomic library was developed by Genetic Identification Services1. Briefly, the DNA was partially digested with a cocktail of seven blunt-end restriction enzymes (HaeIII, StuI, EcoRV, ScaI, BsrBI, PvuII, HiuCII). Fragments between 350 and 700 bp were selected by gel extraction and ligated to a 20 bp oligonucleotide adaptor containing a Hind III restriction site at the 5' end. Microsatellite enrichment was achieved using streptavidin-coated magnetic beads and 5`-biotinylated CA15, AAC12, TACA8 and TAGA8 oligonucleotide probes. The captured molecules were amplified by PCR using a primer complementary to the adaptor, digested with HindIII to remove the adaptor, and ligated into the HindIII site of the pUC19 vector. The plasmids were then electroporated into Escherichia coli DH5. Recombinant clones, identified by blue-white selection, were chosen arbitrarily for sequencing on an ABI 377 using the Big Dye Terminator Cycle Sequencing methodology (Applied Biosystems). Specific primers flanking the identified microsatellite sequences were designed using Designer PCR version 1.03 (Research Genetics) (Table 1). The microsatellite loci were amplified in 10 µl reactions containing 1X PCR buffer, 2 mM MgCl2, 0.2 mM forward primer (fluorescently labeled), 0.2 mM reverse primer, 0.2 mM dNTPs, 0.1 U µl-1 Taq DNA polymerase (Invitrogen), and 20 ng of genomic DNA template. PCR was performed in a PTC-200 (MJ Research) thermal cycler with the following parameters: 94ºC for 3 min, followed by 34 cycles of 94ºC for 40 s, 56ºC for 40 s, 72ºC for 30 s, and a final extension at 72ºC for 4 min. All loci successfully amplified under the same conditions. The PCR products were analyzed on an ABI 3330 DNA sequencer. Alleles were scored using Peak Scanner v1.0, with GS500 (Applied Biosystems) as the internal size standard.

To characterize the polymorphism of each locus we used 100 individuals from Clavelito, Chile. To evaluate the potential presence of null alleles and genotyping artifacts (stutter bands or large dropout alleles) we first checked the data set with MICRO-CHEKER v2.2.3 software (Van Oosterhout et al. 2004). The number of alleles (Na), the expected (He) and observed (Ho) levels of heterozygosity were obtained using GENALEX v6 (Peakall & Smouse 2006). Deviations from Hardy-Weinberg equilibrium (H&W) and gametic disequilibrium between markers were tested using ARLEQUIN v3.1 (Excoffier et al. 2005). All probability values were estimated using 10000 permutations.

RESULTS AND DISCUSSION

We observed a significant departure from HWE in four loci (i.e., OmimA2, OmimB111, OmimC1 and OmimC122). None of the studied loci showed gametic disequilibrium. On the other hand, the results did not show any score errors like large allelle dropout and stutter bands. Two loci showed heterozygotes deficiency: OmimA105 and OmimB111 (Table 1) which, in this case, could be attributed mainly to: a) admixture of individuals captured over a large geographical area that may include more than one panmictic unit (Wahlund effect), b) null alleles, as some of the sequences (allele) could not be amplified, the number of heterozygotes is sub estimated, c) imbreeding and d) selection. According to our results, null alleles are the most plausible cause of heterozygotes deficiency at these two loci, as suggested by the excess of homozygotes obtained by the Micro-Checker software. Heterozygosity deficits due to null alleles have been recorded in Octopus species, (e.g., O. vulgaris, Casu et al. 2002, Cabranes et al. 2008, O. maorum, Doubleday et al. 2008, O. maya, Juárez et al. 2010). The presence of heterozygosity deficit could support the low variability recorded in other molecular markers used in cephalopods (Allozyme, Maltagliati et al. 2002; mitochondrial DNA, Oosthuizen et al. 2004). Moreover, life history traits like semelparity, low fecundity and territorial behavior could promote inbreeding and consequently the heterozygosity deficit.

The Na per locus ranged from 4 to 28, the Ho from 0.273 to 1 and the He varied from 0.651 to 0.946 (Table 1). Polymorphism found in microsatellite loci of O. mimus is comparable with other studies in other members of the genus Octopus. He in O. mimus (mean He = 0.823) was less than He in O. vulgaris (mean He = 0.874, Cabranes et al. 2008; mean He = 0.91, Murphy et al. 2002) and O. maorum (mean He = 0.85, Doubleday et al. 2008). On the other hand, average He in O. mimus was greater than He in O. maya (mean 0.645, Juárez et al. 2010) and O. vulgaris (mean He = 0.765, Greatorex et al. 2000).

In summary, this work provides a significant genetic tool for future population genetic studies, which should improve government management policies to maintain the sustainability of these important fisheries, and helping not only to promote the conservation of the fishing stock, but also to conserve genetic diversity of this cephalopod along the Chilean coast.

ACKNOWLEDGMENTS

Sandra Ferrada and Cristian B. Canales-Aguirre were supported by Doctoral Fellowships for the `Programa de Doctorado en Sistemática y Biodiversidad', from the graduate school of the Universidad de Concepción. Sandra Ferrada was supported by a CONICYT doctoral fellowship. This work forms part of the FIP 2008-39 Project.